The analytical determination of creatinine in urine, serum, or plasma is a widely used and extremely important clinical test for renal dysfunction. It is generally accepted that creatinine clearance is the best measure of glomerular filtration rate. Creatinine levels are also known to vary as a function of disorders such as muscular dystrophy and hypothyroidism.
The presence and concentration of creatinine in these body fluids is most frequently determined by the Jaffe reaction, in which creatinine reacts with picric acid in the presence of excess hydroxide to produce a red color. Many variations of the method have been developed to minimize the effects of numerous interferring materials which are known to be present in real samples.
In the Jaffe-derived procedures for the determination of creatinine, it is generally recommended that the picric acid and sodium hydroxide reagent solutions be stored separately and added individually to the solution to be tested just before the creatinine test, or if the picric acid and sodium hydroxide solutions are premixed to form the alkaline picrate reagent, that this solution should be used immediately or within a very short time. This is because the mixed alkaline picrate solution has a short shelf life. Examples of typical literature statements in this regard are the following, which clearly demonstrate that in the Jaffe alkaline picrate reaction for the determination of creatinine, regardless which of the many variations of the method are employed, stability of the alkaline picrate reagent has been a continuing problem.
a. In the laboratory manual entitled "Physiological Chemistry," published by the United States Naval Medical School in 1951, it is stated on page 170 that the alkaline picrate solution "must be made just prior to use and discarded after standing one hour."
b. In "Practical Clinical Chemistry" by Toro and Ackermann, the procedure for serum states "to each tube add 1 ml of picric acid solution, mix, and add 1 ml of the sodium hydroxide solution."
c. In "Quantitative Clinical Chemistry," Volume 2, by Peters and Van Slyke, Chapter XV, it is stated on page 604, "to the blood filtrate add 5 cc and to the standard solution 10 cc of the freshly prepared alkaline picrate."
d. In "Bray's Clinical Laboratory Methods," revised by Bauer, Ackermann, and Toro, published by the C. V. Mosby Company in 1968, it is stated on page 315, "to each tube add 1 ml picric acid and 1 ml sodium hydroxide solution. Mix, and after standing at room temperature for 20 minutes, read in a photometer."
e. In the article by Shoucri and Pouliot in "Clinical Chemistry," 23, p1527 (1977), it is stated on page 1527 that the mixed alkaline picrate reagent is stable for one day.
f. In the U.S. Pat. No. 4,111,657 by Denney et al, issued in 1978, it is stated in column 6, lines 7-12 that the mixed alkaline picrate reagent has a demonstrated stability in excess of five days when stored at room temperature and protected from light.
g. In the article by Kammeraat, in "Clinica Chimica Acta," 84, p119 (1978), it is stated on page 121 that the mixed alkaline picrate reagent can be used 30 minutes after mixing and is stable for at least one week at room temperature in the dark.
h. In a letter by Jury and Ward in "Clinical Chemistry," 25, p1674 (1979), a recipe for an alkaline buffer containing sodium hydroxide and several other chemicals is given. This buffer is mixed with saturated picric acid solution to prepare a working creatinine reagent. However, in a later communication by Schifreen, Sindab, Bologna, Cameron, and Burnett, in "Clinical Chemistry," 27, p196 (1981), it is stated that a white precipitate forms after about one week of storage of this reagent, making it unusable. These authors recommend adding diethylamine to the mixture to prevent formation of this precipitate for at least one month.
i. In the "Proposed Selected Method" submitted by Haeckel and Hannover, published in "Clinical Chemistry," 27, p179 (1981), it is stated on page 179 that the mixed alkaline picrate solution is stable for at least three months if stored in a dark bottle.
j. In "Clinical Laboratory Methods" by John Bauer, ninth edition, published by the C. V. Mosbey Company in 1982, it is stated on page 490 that the picric acid reagents are mixed just before use.
k. In the communication by Yatzidis, Frangos-Plemenos, and Koutsicos, published in "Clinical Chemistry," 28, p384 (1982), it is stated that the alkaline picrate reagent is prepared by mixing picric acid solution with sodium hydroxide solution just before use.
l. In the article by Bromberg, Pollard, Cheng, and Romaschin, published in "Clinical Chemistry," 30, p281 (1984), it is stated on page 281 that the Technicon RA 1000 creatinine reagent can only be used for four hours after preparation, and that the Beckmann creatinine reagent is stable for one week.
m. In the communication by Posner published in "Clinical Chemistry," 30, p1105 (1984), it is stated with regard to the above-listed publication by Bromberg that the correct statement with regard to the Beckmann creatinine reagent is "the Beckmann alkaline-picrate reagent is a special purpose solution . . . from date of preparation for use, working reagent is stable for 30 days at ambient temperature."
This well documented instability of the alkaline picrate reagent solution is not to be confused with the relatively good stability of picric solutions, either as picric acid or as sodium picrate produced by neutralization of an initial picric acid solution. The alkaline picrate solutions which are the subject of the present invention always contain a substantial molar excess of base relative to the picric acid employed.
In view of the instability of alkaline picrate solutions and the concomitant necessity for preparing such solutions as needed, or at least with some frequency, it would be very desirable to have stable alkaline picrate solutions available. Such stable solutions would eliminate the need to prepare fresh reagent regularly, thereby saving substantial analytical time, and would also assure maximum reproducability of analytical results.